Unraveling the structural secrets of nitric oxide synthases (NOSs) has become an important goal for the purpose of understanding how they can be differentially regulated and/or inhibited by specific substrates or inhibitors. Rat cerebellar NOS, in its cloned, expressed forms, is the subject of this proposal. The formation of L-citrulline and NO. from the amino acid, L-arginine, requires reducing equivalents from NADPH in an overall reaction requiring two monooxygenation steps catalyzed by a single enzyme. Neuronal NOS has a molecular mass of approximately 160 kDa and is expressed in a variety of cell types in brain tissue, as well as in other tissues, such as the gastrointestinal system. The experimental hypothesis is that this highly complex protein, which contains FAD, FMN, and Fe- protoporphyrin IX, as prosthetic groups, and tetrahydrobiopterin and calmodulin binding sequences in a single polypeptide chain, is comprised of domains which have combined in a gene fusion process and exhibit independent folding properties. To examine this hypothesis, the following experimental aims are planned: 1. To demonstrate the existence of independently folding domains of cerebellar nitric oxide synthase, several types of experiments will be performed: a. Microdissection molecular cloning methods, in which the putatively independent domains of neuronal NOS (nNOS) are expressed in E. coli, will continue to be pursued in order to obtain sufficient quantities of material to characterize each of them by biophysical techniques. Preliminary and published data are presented to show the potential for success of this approach. b. Having successfully expressed the N- and C- termini, which represent the heme- and flavin-binding domains of nNOS, respectively, and the dihydrofolate reductase (DHFR) motif in E. coli in ongoing experiments, the PI will attempt the expression of other subdomains. For example, attempts to express the N-terminus of nNOS, which is not present in either the inducible macrophage-type isoform of the constitutive endothelial isoform, will be made. Also, the role of a putative "inhibitory polypeptide", identified by modeling techniques (as was the DHFR motif) as a unique feature of the Ca+2/calmodulin-inducible isoforms, will be examined through microdissection by molecular cloning and expression. Characterization of these domains will utilize a variety of spectroscopic techniques, including fluorescence and optical absorption, circular dichroism (CD), and electron paramagnetic (EPR) and nuclear magnetic resonance (NMR) spectroscopy. 2. To determine how the various domains are folded in three-dimensional space by modeling the structures of those domains which bear significant homology to sequences of enzymes for which there is structural information from X-ray crystallography or 2D NMR. Preliminary data are presented to show structural homologies of various dihydrofolate reductases and several aromatic amino acid hydroxylases with a sequence in the neuronal NOS. These comparisons of modeled structures will be used to determine targets for site-directed mutagenesis and deletion mutations. The wild type and mutated domains will also be compared by fluorescence, absorbance, EPR, and NMR spectroscopy when applicable. NMR techniques can yield 3D structural information on the smaller domains. 3. To attempt, under a variety of conditions, the reconstitution of the form ad on of NO. and L-citrulline from the isolated purified domain fragments. The relationship of the activity and structure of the independently expressed modules to the activity and structure of the intact holoenzyme will be determined.